A Comprehensive Comparison of Clinical Presentation and Outcomes of Kidney Transplant Recipients with COVID-19 during Wave 1 versus Wave 2 at a Tertiary Care Center, India

Data comparing the clinical spectrum of COVID-19 in kidney transplant recipients (KTRs) during the first and second waves of the pandemic in India is limited. Our single-center retrospective study compared the clinical profile, mortality, and associated risk factors in KTRs with COVID-19 during the 1st wave (1st February 2020 to 31st January 2021) and the second wave (1st March-31st August 2021). 156 KTRs with PCR confirmed SARS-CoV-2 infection treated at a tertiary care hospital in New Delhi during the 1st and the second waves were analyzed. The demographics and baseline transplant characteristics of the patients diagnosed during both waves were comparable. Patients in the second wave reported less frequent hospitalization, though the intensive care unit (ICU) and ventilator requirements were similar. Strategies to modify immunosuppressants such as discontinuation of antinucleoside drugs with or without change in calcineurin inhibitors and the use of steroids were similar during both waves. Overall patient mortality was 27.5%. The demographics and baseline characteristics of survivors and nonsurvivors were comparable. A higher percentage of nonsurvivors presented with breathing difficulty, low SpO2, and altered sensorium. Both wave risk factors for mortality included older age, severe disease, ICU/ventilator requirements, acute kidney injury (AKI) needing dialysis, Chest Computerized Tomographic (CT) scan abnormalities, and higher levels of inflammatory markers particularly D-dimer and interleukin-6 levels. Conclusions. KTRs in both COVID-19 waves had similar demographics and baseline characteristics, while fewer patients during the second wave required hospitalization. The D-dimer and IL-6 levels are directly correlated with mortality.


Study Design and Population.
A retrospective study on the effect of COVID-19 on KTRs in India, between the study period 1 st February 2020 and 31 st August 2021, was conducted at a tertiary care hospital in New Delhi, India. 156 KTRs (154 living and 2 deceased donors) identified with SARS-CoV2 real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmed infection and treated as either out-patient or hospitalized were included in the analysis. e two waves of COVID-19 in India, the first wave from 1 st February 2020 to 31 st January 2021 and the second wave from 1 st March 2021 till 31 st August 2021, were analyzed separately. e study evaluated the clinical symptoms, risk factors, laboratory profile, disease management, and mortality rate in KTRs. e present study is a retrospective post-COVID-19 kidney transplant recipient pooled data analysis which excludes any compromise of personal or medical information of the subject. e study was approved by the designated institutional authority of the host institution, Indraprastha Apollo Hospital, New Delhi, to carry out data analysis and publication of manuscript/manuscripts.

Clinical Management of COVID-19 in KTRs.
Treatment and follow-up of all patients were according to the hospital's clinical protocol. COVID-19 infection was diagnosed as per the guidelines of the WHO [55,56]. Patients with positive SARS-CoV-2 RT-PCR from nasopharyngeal or oropharyngeal swabs were considered laboratory-confirmed cases. e disease severity and assessment parameters were as per the Chinese Centre for Disease Control (China CDC) criteria [57]. KTRs with positive SARS-CoV-2 RT-PCR were identified as mild or severe and managed accordingly by a designated COVID-19 treating team in consultation with the treating nephrologist, as described before [34].
Patients were evaluated as per unit protocol ( Figure 1) and were followed up for a minimum of 90 days (except in the case of a fatality).

Data Collection.
Data were collected retrospectively from the medical records of the hospital or patients' followup submissions. Details of any asymptomatic home-isolated patients noncompliant with one or all prescribed drugs or investigation protocols were recorded and included in the study.
Collected data included demographics, transplantation history, comorbidities, concomitant medications, COVID-19-related symptoms, therapy during hospitalization, supportive measures needed during hospitalization, laboratory investigations (other than SARS-CoV-2 RT-PCR), and therapeutic outcomes (mortality and recovery). e onset symptom data were collected on first clinical reporting either

Outcomes.
e primary outcome of the study was to assess the mortality rate associated with COVID-19 in KTRs. e secondary outcomes included the spectrum of clinical presentation, immunosuppressive regimen, laboratory investigations, and pharmacological management of COVID-19 disease in the KTRs and their correlation with ICU admission, AKI, and acquired comorbidities (bacterial, fungal, or viral infections). AKI was defined using the Kidney Disease Improving Global Outcomes (KDIGO)-2012 [59] criteria with baseline serum creatinine. Chest CT scan done in patients with poor oxygen saturation levels regardless of the ongoing treatment was quantified based on the CT severity score index [60].

Statistical Analysis.
e data was analyzed as described before [34]. Briefly, statistical analysis was performed on pooled data tabulated using Microsoft Excel, using the Statistical Package for Social Science (SPSS) version 16.0 (SPSS Inc., Chicago, IL). Continuous variables are represented as mean ± standard deviation (SD), and median and interquartile range (IQR) and qualitative variables are reported as numbers and percentages. For normally distributed variables, mean difference and 95% confidence intervals (CIs) were reported. For skewed variables, the median difference and its 95% CI were calculated using the Hodges Lehmann method; R-software version 3.6.1 was applied for determining the same.
Unpaired Student's t-test was performed to compare the mean between survivor and nonsurvivor groups for normally distributed variables having homogeneity of variance.
e Welch test was applied when the homogeneity of variance between the groups was violated. For inflammatory markers and some biomarkers, the nonparametric Mann-Whitney U test was applied due to skewed distribution. e Chi-square and Fisher's exact test were applied to find the association between mortality and qualitative variables; the odds ratio and its 95% CI were reported.
To compare the discriminate power of biomarkers, Receiver Operating Characteristic (ROC) curve was applied. Multivariable logistic regression (MLR) to find the independent risk factors for nonsurvivors could not be performed due to the small number of cases, and some of the variables had zero count. MLR was performed to evaluate the independent effect of each biomarker on survivor status, adjusting age, hemoglobin (Hb), total leucocyte count (TLC), platelet count, blood urea, albumin level, fungal infection, chronic allograft dysfunction, and CAD/PVD. Bonferroni correction was applied, keeping into consideration the small sample size and multiple variable testing. e p value of less than 0.001 was considered statistically significant.   immunosuppressive regimens of the KTRs diagnosed during the 1 st wave or the 2 nd wave of the COVID-19 pandemic. Similarly, the mean BMI value was also comparable between the two waves. Notably, during both periods, the male to female ratio was skewed toward the male population; however, the difference between the gender distributions was more pronounced in the 2nd wave with male patients (Table 1).  Immunosuppressive treatment regimens were modified in the majority of patients during both waves. In 128 (82.0%) patients, antinucleoside drugs were stopped, whereas in 5 (3.2%) patients, the dose was reduced, and in 19 (12.1%) patients, the treatment was continued as before; four patients (2.6%) were not taking antinucleoside drugs, to begin with. e antinucleoside drugs were stopped in more patients during the second wave than during the first wave (n � 75/84, 89.3% vs n � 53/72, 73.6%, p value 0.011).

Clinical Presentation
CNIs remained unchanged in most patients (n � 116, 74.4%), and the administration was stopped in 22.5% (n � 36) patients; only 2 (1.3%) patients underwent a dose reduction of CNIs. e CNI drug treatment was altered in more patients during the first wave; however, the difference was not statistically significant.

Mortality in COVID-19-Infected KTRs and Comparison of Risk Factors for Mortality in the Two Waves.
e overall patient mortality rate observed was 27.5% [95% CI: 20.7-35.2] (43/156). A detailed comparison of the demographics, immunosuppression regimen, clinical profile, treatment, clinical outcomes, and possible risk factors for mortality between survivors and nonsurvivors is summarized in Tables 4 and 5.
No significant difference was observed between survivors and nonsurvivors with regard to gender, blood group, BMI, and comorbidities (Table 4).
e impact of each biomarker on the survival status of KTRs as evaluated by multivariate logistic regression (MLR) analysis is summarized in Table 6. Only D-dimer and IL6 rise correlated with an increase in mortality; interestingly, every 5-unit increase in IL6 level increased the odds of mortality risk by 2.4% (Table 6).
Additional Receiver Operating Characteristic (ROC) curves were performed to determine the diagnostic values of inflammatory markers; all inflammatory biomarkers were found to be significant for diagnostic purposes (Figure 2). Furthermore, the area under the curve (AUC) was calculated to compare the different classifiers. For purposes of medical diagnosis, AUC values between 0.9 and 1, 0.8-0.9, 0.7-0.8, 0.6-0.7, and 0.5-0.6 were considered excellent, good, fair, poor, and failed, respectively [61]. Based on this classification, IL-6 and CRP were the most acceptable diagnostic markers, followed by procalcitonin and D-dimer ( Figure 2).  e demographics (age, weight, height, BMI, and blood group distribution) of the patients were comparable between the two waves. Interestingly, as reported previously [34], we did observe a male predominance in the patient cohort, the difference being more pronounced in the 2 nd wave.

Discussion
No significant differences in terms of comorbidities, oxygen/ventilator requirement, ICU stay, the incidence of   International Journal of Nephrology AKI (with or without the need for dialysis), and chest CT severity score index were observed between the first and the second wave cohorts. Similar to a previous report [25], we observed more mild COVID-19 cases that did not require hospitalization during the second wave. e baseline immunosuppressive regimens comprising steroids and CNI were comparable between the patients from both waves. e consensus regarding the susceptibility of KTRs in developing severe COVID-19 is that immunosuppressive treatment impairs the immune response [19,24,28]. Treatment guidelines documented in the literature suggest modification of immunosuppression for better COVID-19 management [62]. In our study, immunosuppressive treatments were modified in the majority of patients during both waves in conjunction with other treatment options [63]. e antinucleoside drugs were stopped in more patients during the second wave (89.3% vs 72%) whereas CNI drug treatment was altered for more patients during the first wave.
According to a recent study [25], the use of HCQS and tocilizumab decreased, and that of dexamethasone and remdesivir increased during the second wave. Although not statistically significant, we also observed a decrease in the use of tocilizumab, convalescent plasma, and antibiotics and an increase in treatment with ivermectin and doxycycline during the second wave. Prescriptions for antivirals favipiravir and fluvoxin were administered only during the second wave. e variation between the preferred treatment options in our study and the previous report [25] could be explained by the difference in the patient cohort; our study population included both domiciliary and hospitalized patients while the previous study [25] mainly focussed on hospitalized patients. e modification in treatment made during the second wave could be a result of recent studies demonstrating the efficacies of investigational treatments or drugs that were tried during the first wave, and the introduction of new therapies, thereby providing empirical data for deciding which treatment to follow [1].
During the second wave of COVID-19, India was challenged with the emergence of coronavirus disease-associated mucormycosis in both active and recovered patients, which contributed significantly to the increase in morbidity and mortality of COVID-19 [54,64]. In the present study, during the 2 nd wave, 3 KTRs with COVID-19 developed mucormycosis, while during the 1 st wave, only one patient reported the same. Notably, even though the overall use of antifungal drugs was comparable in the two waves, documented culture positive fungal infections were higher among nonsurvivors (p � 0.015).
In our study, we observed an overall patient mortality rate of 27.5% (43/156), similar to results reported previously by us [34] as well as studies conducted across several countries [12, 19, 21-23, 28, 30, 45, 65-70]. Mortality was significantly associated with ventilator requirement, ICU admission, the incidence of AKI, and the requirement of dialysis support. e distribution of survivors and nonsurvivors as to gender, BMI, comorbidities, and blood group was comparable; however, as reported previously [34], the frequency of nonsurvivors with blood group A was higher (14/66; 38.8%). Studies have shown that individuals with blood group A were susceptible to developing the disease with unfavorable outcomes [71][72][73], possibly due to a lack of anti-A antibodies that have been shown to provide protection against SARS-COV-2 viral infection [74]. Additionally, the blood group A is linked to higher susceptibility of comorbidities that contribute to high mortality of severe COVID-19 [75,76].
Notably, the nonsurvivors significantly presented breathing difficulty with low SpO2 and altered sensorium, supporting that KTRs with severe COVID-19 presentation were more susceptible to mortality.
Previous studies [12, 19, 21-23, 28, 30, 45, 65-70] have reported older age, anemia, low platelet count, higher total leucocyte count, kidney dysfunction as diagnosed by elevated serum creatinine, and blood urea, and CT score >15 and increased inflammatory markers (IL-6, procalcitonin, D-dimer, CRP, Ferritin, and LDH) as risk factors for mortality, which are also present in our study. Our analysis    [21,25,45,47], detailing the similarities and differences between the work, is presented in Table 7. Despite the differences in geographical location and timeline of the epidemic waves, our study presents multiple similarities with other studies, especially with regard to milder symptoms and less hospitalization and COVID-19 treatment strategies during the second wave.
In summary, our study here provides a comprehensive comparison of the effect of COVID-19 on KTRs during the first and second waves of the disease outbreak in India, with relevance to mortality and risk factors associated with it. e inclusion of both hospitalized and home-isolated patients with milder symptoms in the total patient cohort allows us to provide a broader implication. In addition, the extended study period for the 2 nd wave (until 31 st August 2021) permitted us to include patients during the peak and remission of the second wave of the COVID-19 pandemic, thereby providing an inclusive patient cohort for analysis. Our main limitation is that the study is a single-center study with a limited number of participants from a specific geographical location and therefore may not be sufficient to correctly represent the profile of the entire nation. Recent studies have also evaluated the effect of COVID-19 complications such as mucormycosis [54,64], which adds more complexity to the treatment of KTRs. A multicentre study with a larger patient cohort, including a follow-up to study post-COVID-19 consequences, may not only validate our findings for the entire country but also promote awareness for better diagnosis and early management of post-COVID-19 complications.

Conclusions
In our patient cohort, combining both domiciliary and hospitalized individuals, we observed that the demographics and baseline transplant characteristics including the immunosuppressant regimen, comorbidities, requirement of ICU or ventilator, and incidence of AKI and radiological assessment by chest CT scan were similar between both waves. Interestingly, patients in the second wave reported less frequent hospitalization. Immunosuppressant treatments were modified during both waves as a strategy to build an immune response against the SARS-CoV-2 virus and treatment with antivirals favipiravir and fluvoxin was introduced in the second wave. Clinical symptoms such as breathing difficulty, low SpO2, and altered sensorium were presented at a higher rate in nonsurvivors. Common risk factors associated with mortality included older age, severe disease, ICU/ventilator requirements, acute kidney injury (AKI) needing dialysis, CT scan abnormalities, and higher levels of inflammatory markers particularly D-dimer and IL6 levels that correlated directly with mortality. Larger studies are needed to properly assess the outcomes of the second wave among KTRs and to address the potential use of IL6 and D-dimer as diagnostic biomarkers in identifying KTRs with severe COVID-19 disease.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon request.
Ethical Approval e study was approved by the Director Medical Services office of the host institution, Indraprastha Apollo Hospital, New Delhi, to compile and analyze data and publish manuscript/manuscripts.

Conflicts of Interest
ere are no foreseen conflicts of interest associated with this manuscript.